The most common test of acute toxicity is the LD50 test. LD50 means, if administered dose of drugto animal group, for experimental purpose for the estimation of therapeutic effectiveness kills50% of animals, than it means that particular dose of drug is lethal dose 50 (LD50). It wasdeveloped in 1920’s and called “classical LD50” involved 100 animals for 5 dose-groups, laterin 1981 it was modified by the Organization for Economic Co-operation and Development(OECD) and reduced number upto 30 for 3 dose-groups. Methods to calculate LD50 values are -Litchfield and Wilcoxson, Reed-Muench, Miller-Tainter and Karber’s method. But all thesemethods require large number of animals. Factors which affect the results of LD50 are- Species,Age, Sex, Amount of food, Social environment etc. LD50 study has some Limitations and resultsmay vary greatly. Due to excess of animal sacrifice we should go to alternative methods whichminimum number of animals is required. FRAME (Fund for the Replacement of Animals inMedical Experiment) believes that the lethal dose test is unnecessarily cruel and scientificallyinvalid. Several countries, including the UK, have taken steps to ban the oral LD50. The OECD,the international governments’ advisory body abolished the requirement for the oral test in 2001.Three alternative methods and these are: Fixed Dose Procedure (FDP)-OECD TG 420, AcuteToxic Class method (ATC)-OECD TG 423, Up-and-Down Procedure (UDP)-OECD TG 425.These methods only consider signs of toxicity in place of death. Signs recorded during studieslike; increased motor activity, anaesthesia, tremors, arching and rolling. Alternative methodssave numbers experimental animals.______________________________________________________________________________

INTRODUCTION

Toxicity test examine toxic effects when a chemical is absorbed into the body, via mouth, skin,lungs. The most common test of acute (short-term) toxicity is the LD50 test. Many differentsubstances are tested in this way, including all drugs, agricultural chemicals, cleaners some

450Deora Paramveer S., et al J. Chem. Pharm. Res., 2010, 2(6):450-453______________________________________________________________________________cosmetics and their ingredients1. LD50 means if we administer any dose of drug to animal groupfor experimental purpose for the estimation of therapeutic effectiveness of that drug, and if 50%of animal get died than it means that particular dose of drug is lethal dose 50 (LD50). The smallerthe LD50 value, the more toxic is chemical. The opposite is also true: the larger the LD50 value,the lower the toxicity. It was developed in 1920’s and called “classical LD50” involved 100animals for 5 dose-groups, later in 1981 it was modified by the Organization for Economic Co-operation and Development (OECD) and reduced number upto 30 for 3 dose-groups. In 1987further reduced to 20 animals2. Mice, rats, rabbits, guinea pigs, cats, dogs, fish, monkeys andbirds are use for LD50 study3. The LD50 values of a new drug are determined by various route ofadministration (intravenous, intraperitoneal, subcutaneous and oral)4. Results of LD50 study mayaffected by some factors which are - Species, Age, Sex, Amount of food, Social environment,Route of exposure* (oral, dermal, inhalation) and Physical environment such as temperature andhumidity.*Rout of exposure (example, some LD50s for Dichlorvos, an insecticide commonly used inhousehold pesticide strips): - • Oral LD50 (rat): 56 mg/kg • Dermal LD50 (rat): 75 mg/kg • Intraperitoneal LD50: (rat) 15 mg/kg • Inhalation LC50 (rat): 1.7 ppm (15 mg/m3); 4-hour exposure

There are also some Limitations for LD50 study like:- The LD50 gives a measure of theimmediate or acute toxicity, results may vary greatly, LD50 is not tested on humans, All relationto humans are only a guess. The LD50 test is neither reliable nor useful, because the human lethaldose can't be predicted from animal studies.

FRAME (Fund for the Replacement of Animals in Medical Experiment) believes that the lethaldose test is unnecessarily cruel and scientifically invalid. The test involves giving groups ofanimal doses of a test substance until it kills half of them. Several countries, including the UK,have taken steps to ban the oral LD50 and the OECD; the International governments’ advisorybody abolished the requirement for the oral test in 20015.

To give an idea about alternative methods of LD50 and how to reduce the use of animals in LD50study as much as possible.

EXPERIMENTAL SECTION

There are various methods to calculate LD50 values; like the graphical method, arithmeticalmethod and statistical approach. For research purpose, the most widely used method is Litchfieldand Wilcoxson. For routine practical class work; Reed-Muench, Miller-Tainter and Karber’smethod. For calculating LD50 by any one method, find out the least tolerated (smallest) dose(100% mortality) and most tolerated (highest) dose (0% mortality) by hit and trial method. Oncethese two doses are determined, select at least 05 doses in between the least tolerated and mosttolerated doses, and observe mortality due to these doses. Then apply correction factor to 0% and100% mortality group [for 0% dead = 100 (0.25/n) and for 100% dead = 100x (n-0.25/n), wheren = number of death]. The percentage mortality values are converted to probit values by readingthe corresponding probit units from the probit table. Plot the probit value against log doses andread LD50 value as the dose that corresponds to probit 5.6

451Deora Paramveer S., et al J. Chem. Pharm. Res., 2010, 2(6):450-453______________________________________________________________________________(1) Arithmetical method of Karber method 7The sum of the product was divided by the number of animals in a group and the resultingquotient was subtracted from the least lethal dose in order to obtain LD50 value.LD50 = the apparent least dose lethal to all in a group –a b) /NWhere N = number of animals in each group, a = dose difference and b = mean mortality.

(2) Graphical method of Miller and Tainter7

The observed percentage mortality was converted into probit referring to the probit table. Thevalues thus obtained were plotted against log dose. The LD50 value and its standard error weredetermined from the graph, if the line was straight enough.Table 1: - Comparison of different methods:

Contents Method Karber 7 Method of Miller and Method of Lorke 8

Tainter 7No. of rodents used More than necessary More than necessary AppropriateExpenditure High High AverageAccuracy of results Inaccurate Inaccurate Doubtful

The deletion of the LD50 test from the OECD guidelines was due to three alternative methodsbeing adopted which all involve more humane treatment of the animals and use fewer animalsthan the LD50 test. They record toxicity signs in place of death.5

These three alternative tests are:

(1) Fixed Dose Procedure (FDP) — OECD TG 420.

This method does not use death as an end point, instead it uses the observation of clear signs oftoxicity developed at one of a series of fixed dose levels to estimate the LD50.5

(2) Acute Toxic Class method (ATC) — OECD TG 423

This method does not use death as the only end points, it also uses signs of toxicity in itsstepwise approach to estimating the LD50.Principle: - It is based on the Probit model.Procedure: - The ATC method is a sequential testing procedure using only three animals of onesex per step. Depending on the mortality rate three but never more than six animals are used perdose level. This approach results in the reduction of numbers of animals used in comparison tothe LD50 test by 40–70%. 9

(3) Up-and-Down Procedure (UDP) — OECD TG 425

This method does still use death as an end point, but doses animals one at a time to see if thedose needs to be put up or down to achieve an estimate of the LD50therefore giving the minimumnumber of animals a lethal dose of the test substance.

In the up-and-down procedure, animals are dosed one at a time. If an animal survives, the dosefor the next animal is increased; if it dies, the dose is decreased. Each animal is observed for 1 or2 days before dosing the next animal. Surviving animals monitored for delayed death for a totalof 7 days. 10

RESULTS AND DISCUSSION

During our studies we have observed that available alternative methods are more humane thencruel traditional or classical methods. By following these classical methods we are only treatinganimals very cruelly and not getting fruitful results. Better results can be possible throughalternative methods with less number of animals.

CONCLUSION

My all works showed that there is no need to use animals blindly, when there are some goodalternatives are available. By applying the new alternative methods during Pharmacological worktry to avoid animal sacrifice when without sacrifice proper Pharmacological responses arepossible.